1. Field of the Invention
The present invention relates to the novel use of stilbene compounds to effectively potentiate natural epizootic viral infections of insects as a means of insect control.
2. Description of the Related Art
Although several commercial formulations of entomopathogens (e.g., viruses and bacteria) have been available for many years, their use in agricultural and forestry pest management programs has been rather limited. In general, efficacy of currently available formulations of entomopathogens against pestiferous insects has been considered inadequate due to (1) length of time required to subdue the pest (i.e., unacceptable level of crop-damage may occur before the pest succumbs to factors associated with pathogenic infection) and (2) short residual effectiveness against pest infestation (e.g., stability of entomopathogenic viruses is adversely affected by exposure to ultraviolet solar radiation; Jaques, Can. J. Microbiol., 14: 1161-1163 (1968)).
Doan et al. (J. Insect Pathol., 6: 423-429 (1984)) suggested that greater insect control could occur with the use of viruses if the formulation could be enhanced by the addition of certain adjuvants. Potential virus adjuvants included selected analogs or salts of the stilbene compound, 4,4'-diamino-2,2'-stilbene disulfonic acid. Two such analogs are Calcofluor White and Phorwite (Shapiro et al., published U.S. patent application (USDA), Ser. No. 07/609,848, filed Nov. 7, 1990). Results from several laboratory studies reported by Shapiro et al. demonstrated that Calcofluor White M2R potentiated (as much as 1000-fold) the virulence of the nuclear polyhedrosis virus (NPV) against the gypsy moth larvae, Lymantria dispar (as determined by enhanced LT.sub.50 and LD.sub.50 values). Shapiro et al., however, made no claims or suggestions regarding the use of stilbene compounds as enhancers of temperate or virulent indigenous viruses. In fact, the stilbene compounds employed alone in the bioassays which are reported in the literature caused no gypsy moth mortality. There have been no reports of insect mortality from the application of the stilbene compounds alone.
Although the mode-of-action pertaining to the stilbene's ability to potentiate the virulence of a virus against an insect is still speculative, there are reports in the literature regarding the biological properties of stilbene compounds. Stilbenes, and at least some of their photoproducts, have been shown to irreversibly bind to proteins in wool, silk, bovine serum albumin and apomyoglobin (Holt et al., Aust. J. Biol. Sci., 27: 23-29 and 195 (1974)). It has been demonstrated that certain stilbene compounds can inhibit cellulose and chitin microfibril formation (Roberts et al., J. Cell Biology, 9: 115a (1981); Herth, J. Cell Biology, 87: 442-450 (1980)). The stilbene, Calcofluor White, prevented formation of cellulose microfibrils in Acetobacter xylinum by hydrogen bonding with glucan chains (Haigler et al., Science, 210: 903-906 (1980)) and inhibited chitin synthetase activity in Neurospora crassa (Selitrennikoff, Exp. Mycol., 8: 269-272 (1984)). Shapiro et al. found that another stilbene compound, Phorwite AR, synergized a cytoplasmic polyhedrosis virus (CPV) against gypsy moth larvae. Since CPV multiplies only in the midgut epithelial cells, it has been suggested that the site of action of the brightener was the insect's midgut (Dougherty et al., "Mode of Action of Fluorescent Brighteners as Enhancers for the Lymantria Dispar Nuclear Polyhedrosis Virus (Ld NPV) in the Gypsy Moth," oral presentation, Am. Society for Virology, Colorado State University, Fort Collins, Colo., Jul. 9, 1991). With the exception of some fluid-feeding species, many insects possess a midgut which is lined with peritrophic membrane, which in turn, is comprised of chitin and protein (Chapman, The Insects Structure and Function, p. 46 (Elsevier, N.Y. 1971)).
It is well known in the art that baculoviruses are abundant in the environment and most of them are existing at very low levels of infection or in an inactive state. For example, a study examining the prevalence of nuclear polyhedrosis viruses (Trichoplusia ni (Hubner)) on cabbage revealed that a typical coleslaw serving could contain approximately 10.sup.10 polyhedra per serving. Yet, consumers and insects eat this raw cabbage without undue mortality or disease. Similarly, the gypsy moth nuclear polyhedrosis virus does not appear to be highly and reliably virulent when present in the environment. But applied at high concentrations, the gypsy moth NPV becomes deadly to the insect. Prior entomopathogens with or without the concomitant use of stilbene compounds have focused on applying large lethal concentrations of such viruses to target insects. Therefore, it would be very useful for more effective and safe insect control if the virulence of indigenous insect viruses which are found in nature at cryptic and/or low levels of infection could be potentiated. Furthermore, as a consequence of the instability associated with the prior entomopathogenic viruses in practical use, it is highly desirable to find an inexpensive means for enhancing the latent virulence of the ubiquitous, indigenous insect viruses and inducing epizootic viral infections to effectively control insects.